The small regulatory RNA products of the lin-4 and let-7 genes of C. elegans are microRNAs (miRNAs) that control developmental timing in worm larvae. lin-4 and let-7 function by repressing the translation of specific target messenger RNAs via RNA: RNA antisense base-pairing. lin-4, let-7 and certain other miRNAs are evolutionarily conserved among worms, insects and vertebrates, and in some cases, homologs are expressed in similar profiles in diverse animals. These conserved microRNAs may play similar roles in the post-transcriptional control of gene expression during development of vertebrates and invertebrates. This project is designed to determine the in vivo function of four developmentally regulated, evolutionarily conserved, microRNAs in Drosophila melanogaster, let-7, mir-34, mir-100, and mir-125 (the fly ortholog of lin-4). The let-7, mir-125 and mir-100 genes are clustered in the Drosophila genome, and are up-regulated by ecdysone signaling in conjunction with a major developmental transition in flies, larva-to-adult metamorphosis. By contrast, mir-34 expression is down-regulated by ecdysone at the onset of metamorphosis. The goals of this project are to 1) isolate loss-of-function mutations in let-7, mir-125, mir-100, and mir-34, and analyze any resulting defects in fly development; 2) determine the temporal and spatial patterns of expression of let-7, mir-125, mir-100, and mir-34 during Drosophila development; 3) analyze developmental defects resulting from the over-expression and ectopic expression of these miRNAs; 4) identify messenger RNAs that are antisense targets of let-7, mir-125, mir-100, and mir-34; 5) identify additional evolutionarily conserved miRNA genes that are activated or repressed by ecdysone signaling during Drosophila development. This project will exploit Drosophila genetics to define the biological processes that evolutionarily conserved miRNAs control and will illuminate molecular mechanisms fundamental to animal development, including mammals, and insects significant to human health.